Lytic reagent composition for leukocyte differential analysis

Abstract

A lytic reagent composition and the method of use for differential analysis of leukocytes using flow cytometry are disclosed. The lytic reagent composition includes a short chain alkyl oxyethanol, such as 2-methoxyethanol, 2-ethoxyethanol, 2-propoxyethanol, or 2-isopropoxyethanol, in a sufficient amount to preserve leukocytes; a non-lysing nonionic surfactant as a debris solublizer; and an inorganic buffer to maintain pH of the lytic reagent composition in a range from 9.1 to 10.7. The lytic reagent composition is used to lyse red blood cells and preserve leukocytes, as well as used as the sheath reagent for the focus flow measurement. The lytic reagent composition, when used on a flow cytometric analyzer with multiple angle light scatter and light absorbance measurements, enables differentiation of leukocytes into five subpopulations.

1. The method for differential analysis of leukocytes using flow cytometry comprising the step of using as a lytic reagent composition for differential analysis of leukocytes, the composition comprising:
 (a) a short chain alkyl oxyethanol selected from the group consisting of 2-methoxyethanol, 2-ethoxyethanol, 2-propoxyethanol, and 2-isopropoxyethanol in a sufficient amount to preserve leukocytes;  
 (b) a non-lysing nonionic surfactant as a debris solublizer; and  
 (c) an inorganic buffer to maintain pH of said lytic reagent composition in a range from about 9.1 to about 10.7;  
 wherein said lytic reagent composition enables differentiation of leukocytes into subpopulations by optical measurements.  
 
   
   
     2. A method according to  claim 1 , wherein said inorganic buffer is selected from the group consisting of disodium tetraborate decahydrate, dipotassium tetraborate decahydrate, sodium carbonate, sodium bicarbonate, disodium phosphate dipotassium phosphate, trisodium phosphate, and tripotassium phosphate. 
   
   
     3. A method according to  claim 2 , wherein said inorganic buffer is disodium tetraborate decahydrate in amount sufficient to maintain pH of said lytic reagent from about 9.1 to about 10.7. 
   
   
     4. A method according to  claim 1 , wherein said short chain alkyl oxyethanol is 2-propoxyethanol. 
   
   
     5. A method according to  claim 4 , wherein said 2-propoxyethanol is in a concentration range from about 0.003% to about 1% (w/v). 
   
   
     6. A method according to  claim 1 , wherein said non-lysing nonionic surfactant is one selected from the group consisting of polyoxyethylene nonylphenol having from about 30 to about 60 ethylene oxide groups, polyoxyethylene octylphenol having from 30 to about 60 ethylene oxide groups, polyethoxyethylene phosphates having from 30 to about 80 ethylene oxide groups, and polyoxyethylene-polyoxypropylene block copolymer having from about 30 to about 150 ethylene oxide groups and at least 2 propylene oxide groups. 
   
   
     7. A method according to  claim 6 , wherein said a non-lysing nonionic surfactant is polyoxyethylene nonylphenol having from about 30 to about — 60 ethylene oxide group. 
   
   
     8. A method according to  claim 6 , wherein said polyoxyethylene nonylphenol having from about 30 to about 60 ethylene oxide group is in a concentration range from about 0.003% to about 1% (w/v). 
   
   
     9. A method according to  claim 1 , wherein the lytic composition further includes an antimicrobial. 
   
   
     10. A method according to  claim 1 , wherein the lytic reagent composition for differential analysis of leukocytes consists essentially of:
 (a) 2-propoxyethanol in a sufficient amount to preserve leukocytes;  
 (b) polyoxyethylene nonylphenol having from about 30 to about 60 ethylene oxide group; and  
 (c) an inorganic buffer to maintain pH of said lytic reagent composition in a range from about 9.1 to about 10.7.  
 
   
   
     11. A method according to  claim 10 , wherein said inorganic buffer is selected from the group consisting of disodium tetraborate decahydrate, dipotassium tetraborate decahydrate, sodium carbonate, sodium bicarbonate, disodium phosphate, dipotassium phosphate, trisodium phosphate, tripotassium phosphate and mixtures thereof. 
   
   
     12. A method according to  claim 11 , wherein said inorganic buffer is disodium tetraborate decahydrate. 
   
   
     13. A method according to  claim 10 , wherein said 2-propoxyethanol is in a concentration range from about 0.003% to about 1% (w/v). 
   
   
     14. A method according to  claim 10 , wherein said polyoxyethylene nonylphenol having from about 30 to about 60 ethylene oxide group is in a concentration range from about 0.003% to about 1% (w/v). 
   
   
     15. A method according to  claim 10 , wherein said lytic reagent composition further includes an antimicrobial. 
   
   
     16. A method according to  claim 1 , wherein said lytic reagent composition enables differentiation of leukocytes into five subpopulations by optical measurements. 
   
   
     17. A method according to  claim 16 , wherein said five leukocyte subpopulations include neutrophils, lymphocytes, monocytes, eosinophils and basophils. 
   
   
     18. A method according to  claim 16 , wherein said inorganic buffer is selected from the group consisting of disodium tetraborate decahydrate, dipotassium tetraborate decahydrate, sodium carbonate, sodium bicarbonate, disodium phosphate, dipotassium phosphate, trisodium phosphate, and tripotassium phosphate. 
   
   
     19. A method according to  claim 18 , wherein said inorganic buffer is disodium tetraborate decahydrate. 
   
   
     20. A method according to  claim 16 , wherein said short chain alkyl oxyethanol is 2-propoxyethanol. 
   
   
     21. A method according to  claim 20 , wherein said 2-propoxyethanol is in a concentration range from about 0.003% to about 1% (w/v). 
   
   
     22. A method according to  claim 16 , wherein said non-lysing nonionic surfactant is one selected from the group consisting of polyoxyethylene nonylphenol having from about 30 to about 60 ethylene oxide groups, polyoxyethylene octylphenol having from 30 to about 60 ethylene oxide groups, polyethoxyethylene phosphates having from 30 to about 80 ethylene oxide groups, and polyoxyethylene-polyoxypropylene block copolymer having from about 30 to about 150 ethylene oxide groups and at least 2 propylene oxide groups. 
   
   
     23. A method according to  claim 22 , wherein said non-lysing nonionic surfactant is polyoxyethylene nonylphenol having from about 30 to about — 60 ethylene oxide group. 
   
   
     24. A method according to  claim 23 , wherein said polyoxyethylene nonylphenol having from about 30 to about 60 ethylene oxide group is in a concentration range from about 0.003% to about 1% (w/v). 
   
   
     25. A method according to  claim 16 , wherein the lytic reagent composition further comprises an antimicrobial. 
   
   
     26. A method according to  claim 1 , wherein said optical measurements comprises multiple angle light scatter measurements and light absorbance measurement. 
   
   
     27. A method according to  claim 26 , wherein said multiple angle light scatter measurements includes light scatter measurements at from 0° to about 30°, from about 30° to about 50°, and from about 50° to about 90°. 
   
   
     28. A method according to  claim 26 , wherein said light absorbance measurement is axial light absorbance measurement. 
   
   
     29. A lytic reagent composition for differential analysis of leukocytes consisting essentially of:
 (a) a short chain alkyl oxyethanol selected from the group consisting of 2-methoxyethanol, 2-ethoxyethanol, 2-propoxyethanol, and 2-isopropoxyethanol in a sufficient amount to preserve leukocytes;  
 (b) a non-lysing nonionic surfactant as a debris solublizer; and  
 (c) disodium tetraborate decahydrate in amount sufficient to maintain pH of said lytic reagent composition in a range from about 9.1 to about 10.7.  
 
   
   
     30. A lytic reagent composition for differential analysis of leukocytes consisting essentially of:
 (a) 2-propoxyethanol in a sufficient amount to preserve leukocytes;  
 (b) polyoxyethylene nonylphenol having from about 30 to about 60 ethylene oxide group; and  
 (c) disodium tetraborate decahydrate to maintain pH of said lytic reagent composition in a range from about 9.1 to about 10.7.  
 
   
   
     31. A lytic reagent composition for differential analysis or leukocytes comprising:
 (a) a short chain alkyl oxyethanol selected from the group consisting of 2-methoxyethanol, 2-ethoxyethanol, 2-propoxyethanol, and 2-isopropoxyethanol in a sufficient amount to preserve leukocytes;  
 (b) a non-lysing nonionic surfactant as a debris solublizer; and  
 (c) disodium tetraborate decahydrate to maintain pH of said lytic reagent composition in a range from about 9.1 to about 10.7;  
 wherein said lytic reagent composition enables differentiation of leukocytes into five subpopulations by optical measurements.